Aromatic Cage-Directed Azide-Methyllysine Photochemistry for Profiling Nonhistone Interacting Partners of the MeCP2 Methyl-CpG-Binding Domain.

Methyl-CpG-binding protein 2 (MeCP2) is a canonical reader of DNA methylation and a key chromatin regulator implicated in neurodevelopment and cancer. Beyond DNA binding, MeCP2 recognizes trimethylated histone H3 lysine 27 (H3K27me3) through an aromatic cage within its methyl-CpG-binding domain (MBD). Whether this methyllysine-binding interface also mediates interactions with nonhistone methyllysine-containing proteins remains unknown. Here, we developed an aromatic cage-directed chemoproteomic strategy to capture methyllysine-dependent MeCP2 interactions in cells. By site-specifically incorporating the photocrosslinkable unnatural amino acid 4-azido-l-phenylalanine (AzF) into the MeCP2-MBD, we enabled covalent capture of transient MeCP2-binding partners. Engineered MeCP2 variants efficiently crosslinked to methylated histone ligands, validating preserved methyllysine recognition. Proteomic analysis of crosslinked complexes from human cell lysates uncovered previously unrecognized MeCP2-associated proteins involved in chromatin regulation, RNA processing, translation, and metabolism. Integration with single-cell transcriptomic data revealed coordinated expression of MECP2 and its interacting partners in defined cellular populations. Imaging and chromatin-based analyses further demonstrated nuclear colocalization and shared genomic occupancy with selected interactors at transcriptionally relevant loci. Together, these findings establish the MeCP2 aromatic cage as a versatile interaction interface and expand the functional landscape of MeCP2 beyond DNA methylation reading to encompass chromatin-associated regulatory networks.